Blood performs many functions, all of which being vital. Severe hemorrhage or loss of blood endangers life for the following two main reasons: 1) the drop in circulating blood volume reduces tissue perfusion and produces ischemia; and 2) the reduction in oxygen transport impairs tissue oxygenation and produces hypoxia.
The circulatory system reacts to these changes by producing vasoconstriction, which further aggravates ischemia and hypoxia. Ultimately, alterations of cell metabolism and function develop, which lead to shock and death.
In the context of this patent, a "blood substitute" is not a preparation that can replace blood in all of its functions, but an emergency resuscitative fluid that is capable of performing the following functions.
Restoring blood volume. PA1 Transporting oxygen. PA1 Reducing vasoconstriction. PA1 (1) Toxicity brought about by contamination of hemoglobin with environmental bacterial endotoxins, stromal phospholipids, and non-heme proteins and peptides. PA1 (2) High oxygen affinity of hemoglobin in solution interfering with release of oxygen to the tissues. PA1 (3) Instability of Hb molecule and tendency to extravasation and rapid renal excretion. PA1 (4) Tendency of Hb to autoxidation and generation of non-functional met-Hb and toxic oxygen free-radicals. PA1 (5) Transmission by natural Hb of blood-related diseases, such as hepatitis and AIDS. PA1 (1) Centrifugation and filtration, U.S. Pat. No. 3,991,181 to Coczi. PA1 (2) Toluene extraction, U.S. Pat. Nos. 4,001,200 and 4,001,401 to Bonsen. PA1 (3) Ultrafiltration, U.S. Pat. No. 4,526,715 to Kothe et al. PA1 (4) Ultrafiltration plus acid precipitation, U.S. Pat. Nos. 4,136,093 and 4,336,248 to Bonhard et al. PA1 (5) Ion-exchange chromatography, U.S. Pat. No. 4,100,149 to Meiller. PA1 (6) Zinc precipitation, U.S. Pat. Nos. 4,473,494 and 4,529,719 to Tye. PA1 (7) Crystallization, DeVenuto et al., Journal of Laboratory and Clinical Medicine 89: pp. 509-514 (1977). PA1 (1) The plasma volume-expanding effect is of short duration. PA1 (2) Hb passage through the renal glomeruli generates an osmotic diuretic effect which reduces, rather than sustains, plasma volume. PA1 (3) Hb reabsorption in the renal tubules causes injury to the tubular cells. PA1 (4) Hb passage into the interstitial fluids causes edema and cell injury. PA1 (a) Intermolecular cross-linking or polymerization. PA1 (b) Conjugation of Hb with other molecules. PA1 (c) Intramolecular cross-linking of .alpha. or .beta. chains. PA1 (1) Glutaraldehyde is intrinsically toxic and the potential toxicity of its metabolic byproducts is unknown. PA1 (2) Glutaraldehyde is very reactive and tends to form multiple bridges with various Hb sites, such as .alpha.- and .epsilon.-amino groups and sulphydryl groups. This leads to the formation of unpredictable numbers of molecular species. PA1 (3) Polymerization is difficult to control and appears to continue during storage at 4.degree. C., leading to formation of progressively larger polymers of increased viscosity and oxygen affinity. PA1 (4) Non-specific nature of the cross-linking may still permit the presence of Hb dimers in solution. PA1 (1) Complete purification of Hb. PA1 (2) Preparation and stabilization of a Hb with low levels of met-Hb formation. PA1 (3) Addition of oxygen radical-scavengers (M. Feola et al., "Biocompatability of hemoglobins solutions. I. Reactions of vacular endothelial cells to pure and impure hemoglobins," Artificial Organs, 13(3):209-215, 1989).
This fluid, however, must be free of toxic side-effects, as well as of agents of disease such as bacteria and viruses.
For over 50 years, efforts directed to the development of a blood substitute have focused on hemoglobin (Hb), because this is the only substance capable of picking up enough oxygen from atmospheric air to serve as a physiological oxygen carrier. In addition, hemoglobin exerts the same colloid-osmotic pressure as serum albumin and can, therefore, serve as a plasma volume expander. However, up to the present time these efforts have not been successful due to a number of problems outlined below that have been slow to be recognized and difficult to be resolved.
The problem of toxicity, i.e., the ability on the part of Hb solutions to activate the intravascular coagulation of blood and cause damage to the kidney was the first to be recognized. Rabiner in the 1960's popularized the notion that such toxicity was due to the stroma of red blood cells (fragments of red cell membranes) rather than to Hb. He emphasized the need of a stroma-free hemoglobin. However, this term has over the years belied the fact that a Hb truly free of all stromal elements has not been produced. The toxic factors of the red cell membrane were identified by the present inventor and collaborators as the aminophospholipids phosphatidylethanolamine (PE) and phosphatidyl serine (PS), which normally reside on its cytoplasmic side (M. Feola et al., "Toxic factors in the red blood cell membrane," J. of Trauma, 29:1065-1075, 1989). These compounds have a peculiar affinity for Hb and they are more difficult to remove from a Hb solution than other stromal components. When Hb contaminated with PE and PS is infused into experimental animals such as rabbits and monkeys in significant volumes, e.g., at least 1/3 of the animal's calculated blood volume, it causes a systemic inflammatory reaction characterized by activation of intravascular coagulation and complement, activation of leukocytes and platelets, and development of ischemic-inflammatory lesions in the vital organs (M. Feola et al., "Toxicity of polymerized hemoglobin solutions," Surgery, Gynecology & Obstetrics, 166:211-222 1988; M. Feola et al., "Compliment activation and the toxicity of stroma-free hemoglobin solutions in primates,"Circulatory Shock, 25:275-290, 1988).
A problem that has only recently been recognized is the easy contamination of Hb solutions with environmental bacterial endotoxins. Until the development of the limulus amoebocyte lysate test, the U.S. pharmacopoeia relied on the rabbit pyrogenicity test as the assay for tile detection of endotoxins. However, Hb contaminated with endotoxins at concentrations well below its pyrogenicity level was reported to cause the same kind of toxicity as Hb contaminated with aminophospholipids, since the toxic component of endotoxin is in fact a lipid (lipid A). Bacterial endotoxins can be removed from biological solutions by use of affinity chromatography columns, such as Detoxi-Gel columns (Pierce Chemical Co.). However, these columns cannot remove all the endotoxin present if the starting material contains more than 2 endotoxin units per milliliter, as determined by use of the "quantitative chromogenic limulus test" (QCL-1000, Whittaker M. D. Bioproducts) according to which 1 EU is equal to 0.1 nanograms of bacterial lipopolysaccharide.
Hb must be purified from non-heme proteins and peptides. While no toxicity has been associated with the presence of any particular protein, purification is mandated by the necessity of reducing the immunogenicity of natural Hb solutions. It has also been hypothesized that a peptide is responsible for the vasoconstrictor effect of Hb solutions observed in isolated organs such as the heart and kidney, and isolated arteries. A variety of methods for such purification are known to the art that include the following.
None of the methods are totally satisfactory. The above methods (1)-(4) have intrinsic limitations as to the incapability for completely separating Hb from other proteins while methods (5)-(7) do not lend themselves to large-scale purification.
A problem recognized in the 1970's was the high oxygen affinity of Hb in solution. This is the property that regulates the ability of hemoglobin to pick up oxygen from air in the lungs and release it to the tissues. An expression of this property is the P.sub.50 value or partial tension of oxygen at which Hb is 50% saturated. The lower the P.sub.50, the greater the ability of hemoglobin to bind oxygen, and the more reduced its ability to unload oxygen into tissues. The P.sub.50 of human blood is approximately 28 mm Hg whereas the P.sub.50 of human Hb in solution is approximately 13 mm Hg. The difference is due to the fact that within the red blood cell Hb reacts with 2,3-diphosphoglycerate (2,3-DPG), which reduces the affinity of Hb for oxygen. Outside the red blood cell, that interaction is lost and thus Hb binds O.sub.2 so tightly that it ceases to function as an O.sub.2 carrier. To resolve this problem, Benesch et al. developed a covalent reaction of Hb with pyridoxal-5 '-phosphate, a 2,3-DPG analogue. It was at first hoped that such reaction would both reduce oxygen affinity and stabilize the Hb molecule in tetrameric form. However, this failed to materialize. The present inventor and collaborators showed that bovine Hb in solution has the same P.sub.50 value as human blood, and that its affinity for O.sub.2 was regulated by chlorides rattler than by 2,3-DPG (M. Feola et al., "Development of a bovine stroma-free hemoglobin solution as a blood substitute," Surgery, Gynecology & Obstetrics, 157:399-408, 1983). Considering this favorable property, the large-scale availability of bovine RBCs and the low antigenicity of pure hemoglobin among mammals, there are advantages to the use of bovine hemoglobin as the basis for a blood substitute.
Another problem recognized in the 1970's was the rapid extravasation of hemoglobin with short intravascular persistence. This is generally attributed to a tendency of Hb tetramers, .alpha..sub.2 .beta..sub.2, to dissociate into dimers, 2.alpha..beta., which pass with greater ease through the blood capillaries. It now appears that the surface electric charge of tile protein also plays an important role, with electronegativity and low isoelectric point favoring intravascular persistence. Hemoglobin extravasation has the following several undesirable effects.
The prior art has focused exclusively on the prevention of Hb dimerization. For this purpose, the following three types of Hb modification have been developed so far.
The most widely used of the above methods is the intermolecular cross-linking of Hb with glutaraldehyde disclosed in U.S. Pat. Nos. 4,001,200, 4,001,401, and 4,053,590 to Bonsen et al.; 4,061,736 to Morris et al.; 4,136,093 to Bonhard et al, the entire contents of which are incorporated herein by reference. Intermolecular cross-linking by itself suffers from the various drawbacks listed below.
As an alternative, Hb has been coupled with large-size molecules, such as dextran and hydroxyethylstarch (U.S. Pat. No. 4,064,118), polyethylene or polypropylene glycols (U.S. Pat. No. 4,412,986), inulin (U.S. Pat. No. 4,377,512), and poly-alkylene oxide (U.S. Pat. No. 4,670,417). However, these conjugated hemoglobins have increased oxygen affinity and tend to acquire unfavorable properties peculiar to the coupling substances. Intramolecular cross-linking has been achieved by the use of "diaspirin" esters (U.S. Pat. Nos. 4,529,719 to Tye; 4,598,004 to Walder); and "periodate-oxidized adenosine triphosphate" (o-ATP) (Scannon, F. J., "Molecular modification of hemoglobin", Critical Care Medicine 10:261-265(1982); Greenburg, A. G., and Maffuid, P. W., "Modification of hemoglobin--Ring opened diols", Advances in Blood Substitute Research, Liss, Alan R., New York, pp. 9-17 (1983)). However, the diaspirin-hemoglobins still have short intravascular persistence, with a half-life of 3-4 hours, and the ATP-hemoglobins have been found unsatisfactory due to high levels of met-Hb, high oxygen affinity and short half-life.
Significant progress has been reported by reacting human Hb with pyridoxal-5'-phosphate and glutaraldehyde to yield polymerized pyridoxalated Hb ("poly-PLP-hemoglobin"), i.e., a hemoglobin allegedly with both low oxygen affinity and prolonged intravascular persistence (Moss, G. S., et al., "Hemoglobin solution--From tetramet to polymer," Biomaterials, Artificial Cells and Artificial Organs 16(1-3):57-69(1988); DeVenuto, F. and Zegna, A., "Preparation and evaluation of pyridoxalated-polymerized human hemoglobin", J. Surgical Research 34:205-212(1983)). Pyridoxalation, however, was found to interfere with polymerization so that much of the pyridoxalated Hb would remain unpolymerized, while the polymerized Hb would be non-pyridoxalated. As consequence thereof, after infusion of the solution, the Hb with good O.sub.2 transport function would be rapidly excreted via the kidney, while the Hb remaining in the circulation would be of high O.sub.2 affinity.
Over the past few years, questions have been raised concerning an intrinsic toxicity of hemoglobin. On one hand, experimental observations have been reported of a vasoconstrictor effect of Hb. On the other, Hb tends to autoxidize to met-Hb, i.e., the heme iron oxidizes from the ferrous +2 to the ferric +3 state, generating toxic oxygen free-radicals. In view of this, it has been speculated that Hb may act as a pro-oxidant when infused into the circulation. This would produce the lipoperoxidation of cell membranes and cause injury to cell structures. Both these effects, vasoconstriction and generation of oxygen free-radicals would aggravate rather than alleviate the ischemic-hypoxic injuries caused by hemorrhage. Previous experimental studies by the present inventor and collaborators show that both vasoconstriction and the generation of radicals may be controlled by implementation of the following three steps.
Finally, the administration of native Hb solutions carries the risk of transmitting blood product-transmissible diseases. While bacteria and parasites may be easily removed by filtration or ultrafiltration, viruses represent a more serious problem. Two methods of virus inactivation are known to the art. One is a physical method which consists of pasteurizing hemoglobin in its deoxy-form at 60.degree. C. and pH 7.5 for 10 hours. This method has been found to inactivate model viruses such as sindbis, polio, and pseudorabies viruses as well as the human immunodeficiency virus (HIV) (T. N. Estep et al., "Virus inactivation in hemoglobin solutions by heat," Biomaterials, Artificial Cells & Artificial Organs, 16(1-3):129-134 1988). The other is a chemical method that consists of chloroform treatment (S. M. Feinston et al., "Inactivation of hepatitis B virus and non-A, non-B hepatitis by chloroform," Infection & Immunity, 41:816-821, 1983). Both methods, however, produce significant denaturation of Hb, unless special measures are taken.
Thus, there still exists a need for an improved blood substitute, which is stable, has low oxygen affinity, lacks toxicity and is free from blood-transmissible disease particles.